Optical Apparatus and Method for the Inspection of Nucleic Acid Probes by Polarized Radiation

ABSTRACT

An optical apparatus for the inspection of nucleic acid probes includes: a holder ( 22 ) for housing a chip ( 1 ) for analysis of nucleic acids, containing nucleic acid probes ( 12, 12 ′); a light ( 24 ), for supplying an excitation radiation (W E ) to the holder ( 22 ); and an optical sensor ( 25 ) for detecting images (IMG) of the nucleic acid probes ( 12, 12 ′), when a chip ( 1 ) is housed in the holder ( 22 ). The light source ( 24 ) is configured for polarizing the excitation radiation (W E ) according to a excitation polarization direction (D E ). Furthermore, the apparatus is provided with a sensing polarizing filter ( 27 ), which is arranged so as to intercept a reflected portion (W R ) of the excitation radiation (W E ), directed towards the optical sensor ( 25 ). The sensing polarizing filter ( 27 ) has a direction of the sensing polarization (D S ) transverse to the excitation polarization direction (D E ).

TECHNICAL FIELD

The present invention relates to an optical apparatus and to a methodfor the inspection of nucleic acid probes by polarized radiation.

BACKGROUND ART

As is known, the analysis of nucleic acids requires, according todifferent modalities, preliminary steps of preparation of a sample ofbiological material, of amplification of the nucleic material containedtherein, and of hybridization of individual target or reference strands,corresponding to the sequences sought. Hybridization occurs (and thetest yields a positive outcome) if the sample contains strandscomplementary to the target strands.

At the end of the preparatory steps, the sample must be examined tocontrol whether hybridization has occurred (the so called detectionstep). For this purpose, various inspection methods and apparatuses areknown, for example of an optical or electrical type. In particular, themethods and apparatuses of an optical type are frequently based upon thephenomenon of fluorescence. The reactions of amplification andhybridization are conducted so that the hybridized strands, contained ina detection chamber made in a support, include fluorescent molecules orfluorofors (the hybridized strands may be either grafted to the bottomof the detection chamber or remain in liquid suspension). The support isexposed to a light source having an appropriate spectrum of emission,such as to excite the fluorofors. In turn, the excited fluorofors emit asecondary radiation at an emission wavelength higher than the peak ofthe excitation spectrum. The light emitted by the fluorofors iscollected and captured by an optical sensor. In order to eliminate thebackground light radiation, which represents a source of disturbance,the optical sensor is provided with band-pass or interferential filterscentred at the wavelength of emission of the fluorofors.

However, the difference between the maximum peak of the emissionspectrum of the fluorofors and the peak of the excitation spectrum (alsoreferred to as “Stokes shift”) is not very high, and the filters,however selective they may be, can only attenuate the light emitted bythe source and subsequently diffused, without, however, eliminating italtogether. It should also be taken into account that the materials usedfor providing the supports often have high reflecting power. Forexample, microfluidic devices for the analysis of nucleic acidsintegrated in semiconductor chips are increasingly widespread. Inintegrated microfluidic devices, the detection chamber often has thebottom coated with a layer of silicon dioxide and, sometimes, also metalelectrodes are present, for example of gold or aluminium. In effect,hence, only a relatively small part of the light emitted by the sourceis absorbed, whereas a conspicuous fraction is reflected and ispotentially capable of disturbing the detection of the light emitted bythe fluorofors.

DISCLOSURE OF INVENTION

The aim of the present invention is to provide an optical apparatus anda method for the inspection of nucleic acid probes with polarizedradiation which will be free from the limitations described.

According to the present invention, an optical apparatus and a methodfor the inspection of nucleic acid probes are provided, as defined inClaims 1 and 8, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention, some embodiments thereofare now described, purely by way of non-limiting example and withreference to the attached plate of drawings, wherein:

FIG. 1 is a top plan view of a chip for analysis of nucleic acids;

FIG. 2 is a cross-sectional view through the chip of FIG. 1;

FIG. 3 is a simplified block diagram of an optical apparatus inaccordance with a first embodiment of the present invention;

FIG. 4 is a schematic illustration of the apparatus of FIG. 3 in use,into which the chip of FIGS. 1 and 2 has been loaded;

FIG. 5 is a graph that shows quantities regarding the apparatus of FIG.3; and

FIG. 6 is a simplified block diagram of an optical apparatus inaccordance with a second embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

FIGS. 1 and 2 show a chip 1 in which a chemical microreactor for theanalysis of nucleic acids (here DNA) is provided. The chip 1 comprises:a substrate 2 made of semiconductor material; inlet reservoirs 4; aplurality of microfluidic channels 5; heaters 6 associated to themicrofluidic channels 5; and a detection chamber 7.

More precisely, the inlet reservoirs 4 and the detection chamber 7 aredefined in a structural layer 9 arranged on the surface of the substrate2 (for example, the structural layer 9 may either comprise a resistlayer deposited on the substrate 2 or a glass chip glued thereto).

The microfluidic channels 5 are buried within the substrate 2, forexample as described in EP-A-1 043 770, EP-A-1 130 631, or in thepublished patent application US 2005/282221, and extend between theinlet reservoirs 4 and the detection chamber 7. Furthermore, themicrofluidic channels 5 are fluidly coupled both to the inlet reservoirs4, through inlet openings 10, so as to be accessible from outside, andto the detection chamber 7, through outlet openings 11.

The heaters 6, here including resistive elements made of polysilicon,are formed on the surface of the substrate 2 and extend in a directiontransverse to the microfluidic channels 5. Furthermore, the heaters 6are electrically connectable in a known way to external electric powersources (not shown) and can be driven to release thermal power to themicrofluidic channels 5 so as to control the temperature within themcyclically according to predetermined thermal profiles.

The detection chamber 7 accommodates a plurality of so called “DNAprobes” 12, comprising single stranded reference DNA containingpredetermined sequences of nucleotides. More precisely, the DNA probes12 are arranged in predetermined positions so as to form an array andare grafted to an anchorage layer 14, which forms the bottom of thedetection chamber 7. After a step of hybridization, some of the DNAprobes, designated by 12′, are hybridized, i.e., they are bound toindividual complementary DNA sequences, and contain fluorofors 15.

The microreactor integrated in the chip 1 is prearranged for performingreactions of amplification of nucleic material, for example by PCR(Polymerase Chain Reaction), and hybridization of the DNA probes 12. Forthis purpose, a biological sample containing nucleic material previouslytreated is supplied to the inlet reservoirs 4 and fed into themicrofluidic channels 5. Here, the sample is subjected to thermalcycling in order to amplify the DNA present, in a known way. At the endof the amplification step, the biological sample is further made toadvance as far as the detection chamber 7, where the DNA probes 12 arelocated. If the biological sample contains a sequence of nucleotidescomplementary to the DNA probes 12, the latter are hybridized.Furthermore, the amplification reactions are conducted so that thehybridized DNA probes 12′ will contain fluorofors 15 (shown onlyschematically) having a characteristic emission wavelength.

With reference to FIG. 3, number 20 designates an optical inspectionapparatus for the detection of hybridized DNA strands, based uponfluorescence. The inspection apparatus 20 comprises a control unit 21, aholder 22 for housing an item of the chip 1, a light excitation device24 and an optical sensor 25, provided with a collimation and focusingdevice 26 and a sensing polarizing filter 27. FIG. 3 moreover shows adetail of the chip 1 loaded into the holder 22 so as to be examined.

The light source 24 comprises a radiant element 28, for example anincandescent lamp or a LED, which emits incoherent non-polarizedradiation W₀ with an emission spectrum such as to excite the fluorofors15, and an excitation polarizing filter 30 associated to the radiantelement 28. The excitation polarizing filter 30, of a linear type, ispositioned so as to intercept the non-polarized radiation W₀ emitted bythe radiant element 28 and has a predetermined excitation polarizationdirection D_(E) (i.e., the radiation emerging from the excitationpolarizing filter 30 is polarized according to the excitationpolarization direction D_(E)). Consequently, the excitation radiationW_(E) that leaves the light source 24 is linearly polarized according tothe excitation polarization direction D_(E).

The light source 24 is moreover oriented so that the excitationradiation W_(E) reaches the detection chamber 7 of the chip 1 with apredetermined angle of incidence (for example 45°).

The optical sensor 25, for example of a CMOS or CCD type, receives thefluorescent radiation W_(F) emitted by the fluorofors 15 in thedetection chamber 7 of the microreactor integrated in the chip 1. Moreprecisely, the collimation and focusing device 26 is arranged so as tocollect the fluorescent radiation W_(F) emitted in a directionsubstantially perpendicular to the chip 1 and orient it in the directionof the optical sensor 25.

The sensing polarizing filter 27, which is also of a linear type, ispositioned between the collimation and focusing device 26 and theoptical sensor 25 so as to intercept the fraction of excitationradiation W_(E) coming from the light source 24 and reflected ordiffused by the chip 1 in the direction of the optical sensor 25(reflected radiation W_(R); for reasons of convenience, the term“reflected radiation” will be used hereinafter to indicate both thefraction of radiation incident on the chip 1 that is reflected accordingto a macroscopically predictable path and the fraction of incidentradiation that is diffused in the direction of the optical sensor 25,for example on account of the imperfect homogeneity of the surface ofthe chip 1). The sensing polarizing filter 27 has a sensing polarizationdirection D_(S) that is transverse, preferably perpendicular, to theexcitation polarization direction D_(E) of the reflected radiationW_(R). In this connection (see FIG. 5), the reflection does not modifysubstantially the direction of the electric field E associated to theexcitation radiation (parallel to the excitation polarization directionD_(E)), whereas the direction of propagation K_(R) of the reflectedradiation W_(R) is determined by the surface conformation of the chip 1(as well as, of course, by the direction of propagation K_(E) of theincident excitation radiation), according to the laws of geometricaloptics. A part of the reflected radiation W_(R) is thus directed towardsthe optical sensor 25 along an optical path P. The sensing polarizingfilter 27 is arranged along the optical path P in a plane substantiallyperpendicular to the direction of propagation K_(R) of the reflectedradiation W_(R) directed towards the optical sensor 25. The sensingpolarization direction D_(S) of the sensing polarizing filter 27 isperpendicular to the excitation polarization direction D_(E) (i.e.,perpendicular to the direction of the electric field E associated to thereflected radiation W_(R)). Preferably, moreover, the orientation of thesensing polarizing filter 27 is adjustable so as to achieve the mostcorrect alignment.

The inspection apparatus 20 operates as described hereinafter.Initially, an item of the chip 1, integrating a microreactor in which astep of hybridization of the DNA probes 12 has been performed, is loadedinto the holder 22. The control unit 21 activates the light source 24,and the excitation radiation W_(E) emitted reaches the detection chamber7. A fraction of the excitation radiation W_(E) incident on the chip 1is absorbed by the fluorofors 15 of the hybridized DNA probes 12′,whereas the remaining part is reflected or diffused in variousdirections, according to the surface conformation of the chip 1. Thefluorofors 15 are hence excited and emit in an approximately isotropicway an fluorescent radiation W_(F), which does not preserve the state ofpolarization of the excitation radiation W_(E).

Consequently, a part of the fluorescent radiation W_(F) and thereflected or diffused radiation W_(E) directed towards the opticalsensor 25 reach the sensing polarizing filter 27, which is located infront of the optical sensor 25. The reflected radiation W_(R) isintercepted and almost completely blocked by the sensing polarizingfilter 27, because it is polarized in a direction substantiallyperpendicular to the sensing polarization direction D_(S). Inparticular, the effectiveness of the sensing polarizing filter 27 ishigher the closer the sensing polarization direction D_(S) is to beingperpendicular to the polarization direction of the reflected radiationW_(R) (i.e., the excitation polarization direction D_(E)). Thefluorescent radiation W_(F) due to the excitation of the fluorofors 15,instead, is in part attenuated, but not eliminated completely(transmitted radiation W_(T)), and can hence reach the optical sensor25, which detects an image IMG and sends it to the control unit 21.

Advantageously, the sensing polarizing filter 27 enables practicallytotal elimination of the excitation radiation emitted by the lightsource 24 and reflected by the chip 1, which represents a disturbance.Consequently, the images detected by the optical sensor 25 are producedsubstantially only by the fluorescent radiation and enable detection ofthe hybridized DNA probes 12′ in an extremely reliable way.

A different embodiment of the invention is illustrated in FIG. 6, whereparts that are the same as those already shown are designated by thesame reference numbers. In this case, an optical inspection apparatus 20for the detection of hybridized DNA strands, based upon fluorescence,comprises the control unit 21, the holder 22, a light source 124, theoptical sensor 25, the collimation and focusing device 26, and thesensing polarizing filter 27.

The light source 124 comprises an emitter element 130, which generatesdirectly a coherent monochromatic excitation radiation W_(E), polarizedaccording to a excitation polarization direction D_(E) (for example, alaser emitter). The emission is spontaneously polarized, and the use ofbiasing filters associated to the light source 124 is not required.

The sensing polarizing filter 27 is once again oriented so that thesensing polarization direction D_(S) is substantially perpendicular tothe polarization direction of the reflected radiation W_(R), which is inpractice the excitation polarization direction D_(E).

Hence, as has already been described, the reflected radiation W_(R) isblocked by the sensing polarizing filter 27, and only the fluorescentradiation W_(E) emitted by the fluorofors 15 is transmitted to theoptical sensor 25.

Finally, it is evident that modifications and variations may be made tothe apparatus and method described herein, without departing from thescope of the present invention, as defined in the annexed claims.

1. An optical apparatus for the inspection of nucleic acid probes,comprising: a holder for receiving a chip for analysis of nucleic acids,containing nucleic acid probes; a light excitation device for supplyingan excitation radiation to said holder, wherein said excitationradiation is polarized according to a first polarization direction; anoptical sensor for detecting images of said nucleic acid probes whensaid chip is housed in said holder; and a sensing polarizing filterhaving a second polarization direction transverse to said firstpolarization direction and arranged so as to intercept a reflectedportion of said excitation radiation directed towards said opticalsensor.
 2. The apparatus according to claim 1, wherein said secondpolarization direction is substantially perpendicular to said firstpolarization direction.
 3. The apparatus according to claim 1, whereinsaid light excitation device comprises a radiant element supplying anon-polarized radiation and an excitation polarizing filter forpolarizing said non-polarized radiation according to said firstpolarization direction.
 4. The apparatus according to claim 1, whereinsaid light excitation device comprises a polarized radiation emitterelement.
 5. The apparatus according to claim 4, wherein said polarizedradiation emitter element is a laser emitter.
 6. The apparatus accordingto claim 1, wherein said light excitation device is oriented so thatsaid excitation radiation reaches said chip with an angle of incidenceof approximately 45°.
 7. The apparatus according to claim 1, comprisinga collimation and focusing device arranged so as to collect afluorescent radiation emitted by said nucleic acid probes in a directionsubstantially perpendicular to said chip and to orient said fluorescentradiation collected towards said optical sensor.
 8. A method for theinspection of nucleic acid probes, comprising the steps of: sending anexcitation radiation to a chip containing nucleic acid probes, whereinsaid excitation radiation is polarized according to a firstpredetermined direction; and filtering a reflected portion of saidexcitation radiation with a polarizing filter, wherein said polarizingfilter has a second polarization direction that is transverse to saidfirst polarization direction.